Natriuretic polypeptides with unique pharmacologic profiles

ABSTRACT

This document provides natriuretic polypeptides. For example, this document provides polypeptides having a natriuretic activity. In some cases, a polypeptide provided herein can have natriuretic activities without inducing excessive hypotension. This document also provides methods and materials for inducing natriuretic activities within a mammal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/257,552, filed Apr. 21, 2014, which is a divisional of U.S.application Ser. No. 13/002,178, filed Mar. 2, 2011, which is a NationalStage application under 35 U.S.C. §371 of International Application No.PCTUS2009/047534, filed Jun. 16, 2009, which claims benefit of priorityfrom U.S. Provisional Application Ser. No. 61/077,824, filed on Jul. 2,2008. The disclosures of the prior applications are considered part of(and are incorporated by reference in) the disclosure of thisapplication.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant HL036634awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

1. Technical Field

This document relates to natriuretic polypeptides. For example, thisdocument provides methods and materials related to natriureticpolypeptides having an improved pharmacologic profile (e.g., reducedability to induce severe hypotension) and the use of such natriureticpolypeptides to treat or prevent cardiovascular, renal, or metabolicdisease conditions such as ischemia heart disease, heart failure,hypertensive heart disease, and renal dysfunction.

2. Background Information

Natriuretic polypeptides are polypeptides that can cause natriuresis(increased sodium excretion in the urine). Such polypeptides can beproduced by brain, heart, kidney, and/or vasculature tissue.

SUMMARY

This document relates to natriuretic polypeptides. For example, thisdocument provides methods and materials related to natriureticpolypeptides having an improved pharmacologic profile (e.g., reducedability to induce severe hypotension) and the use of natriureticpolypeptides to treat or prevent cardiovascular conditions, renalconditions, metabolic disease conditions, or combinations thereof. Insome cases, a polypeptide provided herein can have the ability toincrease plasma cGMP levels, the ability to increase urinary cGMPexcretion, the ability to increase urine flow, the ability to increaseurinary sodium excretion, the ability to increase renal blood flow, theability to alter renal perfusion pressure, the ability to reduce renalvascular resistance, the ability to reduce proximal fractionalreabsorption of sodium, the ability to reduce distal fractionalreabsorption of sodium, the ability to increase urinary potassiumexcretion, the ability to reduce mean arterial pressure, the ability toreduce pulmonary capillary wedge pressure, the ability to reduce rightatrial pressure, the ability to reduce pulmonary arterial pressure, theability to reduce systemic vascular resistance, the ability to changepulmonary vascular resistance, the ability to increase hematocrit, theability to reduce plasma renin activity, the ability to reduce plasmaangiotensin II levels, the ability to reduce plasma aldosterone levels,the ability to increase plasma ANP immunoreactivity levels, the abilityto increase urinary excretion of ANP, the ability to increase plasma CNPimmunoreactivity levels, and the ability to increase urinary CNPexcretion. In some cases, a polypeptide provided herein can exert mildeffects on blood pressure. In some cases, a polypeptide provided hereincan be an agonist for natriuretic peptide receptor-A, natriureticpeptide receptor-B, or both natriuretic peptide receptor-A andnatriuretic peptide receptor-B.

In general, one aspect of this document features a polypeptide less than44 amino acid residues in length, wherein the polypeptide comprises, inan order from amino terminus to carboxy terminus: (a) the sequence setforth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:1 with nomore than three additions, subtractions, or substitutions, (b) thesequence set forth in SEQ ID NO:2 or the sequence set forth in SEQ IDNO:2 with no more than five additions, subtractions, or substitutions,and (c) the sequence set forth in SEQ ID NO:3 or the sequence set forthin SEQ ID NO:3 with no more than three additions, subtractions, orsubstitutions. The polypeptide can comprise natriuretic activity. Thepolypeptide can comprise a cGMP-activating property. The polypeptide cancomprise a natriuretic and diuretic activity. The polypeptide cancomprise a cardiac-unloading activity. The polypeptide can lack theability to induce excessive hypotension. The polypeptide can comprisethe sequence set forth in SEQ ID NO:1. The polypeptide can comprise thesequence set forth in SEQ ID NO:2. The polypeptide can comprise thesequence set forth in SEQ ID NO:3. The polypeptide can comprise thesequence set forth in SEQ ID NO:1, the sequence set forth in SEQ IDNO:2, and the sequence set forth in SEQ ID NO:3. The polypeptide cancomprise the sequence set forth in SEQ ID NO:1 with no more than threeconservative amino acid substitutions. The polypeptide can comprise thesequence set forth in SEQ ID NO:2 with no more than five conservativeamino acid substitutions. The polypeptide can comprise the sequence setforth in SEQ ID NO:3 with no more than three conservative amino acidsubstitutions. The polypeptide can be a substantially pure polypeptide.

In another aspect, this document features a polypeptide less than 44amino acid residues in length, wherein the polypeptide comprises, in anorder from amino terminus to carboxy terminus: (a) the sequence setforth in SEQ ID NO:6 or the sequence set forth in SEQ ID NO:6 with nomore than three additions, subtractions, or substitutions, (b) thesequence set forth in SEQ ID NO:2 or the sequence set forth in SEQ IDNO:2 with no more than five additions, subtractions, or substitutions,and (c) the sequence set forth in SEQ ID NO:7 or the sequence set forthin SEQ ID NO:7 with no more than three additions, subtractions, orsubstitutions. The polypeptide can comprise natriuretic activity. Thepolypeptide can comprise a cGMP-activating property. The polypeptide cancomprise a natriuretic and diuretic activity. The polypeptide cancomprise a cardiac-unloading activity. The polypeptide can lack theability to induce excessive hypotension. The polypeptide can comprisethe sequence set forth in SEQ ID NO:6. The polypeptide can comprise thesequence set forth in SEQ ID NO:2. The polypeptide can comprise thesequence set forth in SEQ ID NO:7. The polypeptide can comprise thesequence set forth in SEQ ID NO:6, the sequence set forth in SEQ IDNO:2, and the sequence set forth in SEQ ID NO:7. The polypeptide cancomprise the sequence set forth in SEQ ID NO:6 with no more than threeconservative amino acid substitutions. The polypeptide can comprise thesequence set forth in SEQ ID NO:2 with no more than five conservativeamino acid substitutions. The polypeptide can comprise the sequence setforth in SEQ ID NO:7 with no more than three conservative amino acidsubstitutions. The polypeptide can be a substantially pure polypeptide.

In another aspect, this document features an isolated nucleic acidencoding a polypeptide selected from the group consisting of: (1) apolypeptide less than 44 amino acid residues in length, wherein thepolypeptide comprises, in an order from amino terminus to carboxyterminus: (a) the sequence set forth in SEQ ID NO:1 or the sequence setforth in SEQ ID NO:1 with no more than three additions, subtractions, orsubstitutions, (b) the sequence set forth in SEQ ID NO:2 or the sequenceset forth in SEQ ID NO:2 with no more than five additions, subtractions,or substitutions, and (c) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three additions,subtractions, or substitutions, and (2) a polypeptide less than 44 aminoacid residues in length, wherein the polypeptide comprises, in an orderfrom amino terminus to carboxy terminus: (a) the sequence set forth inSEQ ID NO:6 or the sequence set forth in SEQ ID NO:6 with no more thanthree additions, subtractions, or substitutions, (b) the sequence setforth in SEQ ID NO:2 or the sequence set forth in SEQ ID NO:2 with nomore than five additions, subtractions, or substitutions, and (c) thesequence set forth in SEQ ID NO:7 or the sequence set forth in SEQ IDNO:7 with no more than three additions, subtractions, or substitutions.In another aspect, this document features a vector comprising a nucleicacid encoding a polypeptide selected from the group consisting of: (1) apolypeptide less than 44 amino acid residues in length, wherein thepolypeptide comprises, in an order from amino terminus to carboxyterminus: (a) the sequence set forth in SEQ ID NO:1 or the sequence setforth in SEQ ID NO:1 with no more than three additions, subtractions, orsubstitutions, (b) the sequence set forth in SEQ ID NO:2 or the sequenceset forth in SEQ ID NO:2 with no more than five additions, subtractions,or substitutions, and (c) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three additions,subtractions, or substitutions, and (2) a polypeptide less than 44 aminoacid residues in length, wherein the polypeptide comprises, in an orderfrom amino terminus to carboxy terminus: (a) the sequence set forth inSEQ ID NO:6 or the sequence set forth in SEQ ID NO:6 with no more thanthree additions, subtractions, or substitutions, (b) the sequence setforth in SEQ ID NO:2 or the sequence set forth in SEQ ID NO:2 with nomore than five additions, subtractions, or substitutions, and (c) thesequence set forth in SEQ ID NO:7 or the sequence set forth in SEQ IDNO:7 with no more than three additions, subtractions, or substitutions.

In another aspect, this document features a host cell comprising anucleic acid encoding a polypeptide selected from the group consistingof: (1) a polypeptide less than 44 amino acid residues in length,wherein the polypeptide comprises, in an order from amino terminus tocarboxy terminus: (a) the sequence set forth in SEQ ID NO:1 or thesequence set forth in SEQ ID NO:1 with no more than three additions,subtractions, or substitutions, (b) the sequence set forth in SEQ IDNO:2 or the sequence set forth in SEQ ID NO:2 with no more than fiveadditions, subtractions, or substitutions, and (c) the sequence setforth in SEQ ID NO:3 or the sequence set forth in SEQ ID NO:3 with nomore than three additions, subtractions, or substitutions, and (2) apolypeptide less than 44 amino acid residues in length, wherein thepolypeptide comprises, in an order from amino terminus to carboxyterminus: (a) the sequence set forth in SEQ ID NO:6 or the sequence setforth in SEQ ID NO:6 with no more than three additions, subtractions, orsubstitutions, (b) the sequence set forth in SEQ ID NO:2 or the sequenceset forth in SEQ ID NO:2 with no more than five additions, subtractions,or substitutions, and (c) the sequence set forth in SEQ ID NO:7 or thesequence set forth in SEQ ID NO:7 with no more than three additions,subtractions, or substitutions. The host cell can be a eukaryotic hostcell.

In another aspect, this document features a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a polypeptideselected from the group consisting of: (1) a polypeptide less than 44amino acid residues in length, wherein the polypeptide comprises, in anorder from amino terminus to carboxy terminus: (a) the sequence setforth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:1 with nomore than three additions, subtractions, or substitutions, (b) thesequence set forth in SEQ ID NO:2 or the sequence set forth in SEQ IDNO:2 with no more than five additions, subtractions, or substitutions,and (c) the sequence set forth in SEQ ID NO:3 or the sequence set forthin SEQ ID NO:3 with no more than three additions, subtractions, orsubstitutions, and (2) a polypeptide less than 44 amino acid residues inlength, wherein the polypeptide comprises, in an order from aminoterminus to carboxy terminus: (a) the sequence set forth in SEQ ID NO:6or the sequence set forth in SEQ ID NO:6 with no more than threeadditions, subtractions, or substitutions, (b) the sequence set forth inSEQ ID NO:2 or the sequence set forth in SEQ ID NO:2 with no more thanfive additions, subtractions, or substitutions, and (c) the sequence setforth in SEQ ID NO:7 or the sequence set forth in SEQ ID NO:7 with nomore than three additions, subtractions, or substitutions.

In another aspect, this document features a method for increasingnatriuretic activity within a mammal without excessively lowering bloodpressure. The method comprises administering, to the mammal, apolypeptide selected from the group consisting of: (1) a polypeptideless than 44 amino acid residues in length, wherein the polypeptidecomprises, in an order from amino terminus to carboxy terminus: (a) thesequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ IDNO:1 with no more than three additions, subtractions, or substitutions,(b) the sequence set forth in SEQ ID NO:2 or the sequence set forth inSEQ ID NO:2 with no more than five additions, subtractions, orsubstitutions, and (c) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three additions,subtractions, or substitutions, and (2) a polypeptide less than 44 aminoacid residues in length, wherein the polypeptide comprises, in an orderfrom amino terminus to carboxy terminus: (a) the sequence set forth inSEQ ID NO:6 or the sequence set forth in SEQ ID NO:6 with no more thanthree additions, subtractions, or substitutions, (b) the sequence setforth in SEQ ID NO:2 or the sequence set forth in SEQ ID NO:2 with nomore than five additions, subtractions, or substitutions, and (c) thesequence set forth in SEQ ID NO:7 or the sequence set forth in SEQ IDNO:7 with no more than three additions, subtractions, or substitutions.

In another aspect, this document features a method for treating a mammalhaving a cardiovascular condition or renal condition. The methodcomprises administering, to the mammal, a polypeptide under conditionswherein the severity of a manifestation of the cardiovascular conditionor renal condition is reduced. The polypeptide is selected from thegroup consisting of: (1) a polypeptide less than 44 amino acid residuesin length, wherein the polypeptide comprises, in an order from aminoterminus to carboxy terminus: (a) the sequence set forth in SEQ ID NO:1or the sequence set forth in SEQ ID NO:1 with no more than threeadditions, subtractions, or substitutions, (b) the sequence set forth inSEQ ID NO:2 or the sequence set forth in SEQ ID NO:2 with no more thanfive additions, subtractions, or substitutions, and (c) the sequence setforth in SEQ ID NO:3 or the sequence set forth in SEQ ID NO:3 with nomore than three additions, subtractions, or substitutions, and (2) apolypeptide less than 44 amino acid residues in length, wherein thepolypeptide comprises, in an order from amino terminus to carboxyterminus: (a) the sequence set forth in SEQ ID NO:6 or the sequence setforth in SEQ ID NO:6 with no more than three additions, subtractions, orsubstitutions, (b) the sequence set forth in SEQ ID NO:2 or the sequenceset forth in SEQ ID NO:2 with no more than five additions, subtractions,or substitutions, and (c) the sequence set forth in SEQ ID NO:7 or thesequence set forth in SEQ ID NO:7 with no more than three additions,subtractions, or substitutions. Administration of the polypeptide to themammal can be such that it does not excessively lower the blood pressureof the mammal.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a BAA-NP polypeptide that is 28 aminoacid residues in length (SEQ ID NO:4). The first six amino acid residuesof SEQ ID NO:4 correspond to amino acid residues 1 to 6 of human ANP andare designated as SEQ ID NO:1. Amino acid residues 7 to 23 of SEQ IDNO:4 correspond to amino acid residues 10 to 26 of human mature BNP andare designated as SEQ ID NO:2. Amino acid residues 24 to 28 of SEQ IDNO:4 correspond to amino acid residues 24 to 28 of human ANP and aredesignated as SEQ ID NO:3.

FIG. 2 is a schematic diagram of a BUA-NP polypeptide that is 32 aminoacid residues in length (SEQ ID NO:5). The first ten amino acid residuesof SEQ ID NO:5 correspond to amino acid residues 1 to 10 of humanurodilatin and are designated as SEQ ID NO:6. Amino acid residues 11 to27 of SEQ ID NO:5 correspond to amino acid residues 10 to 26 of humanmature BNP and are designated as SEQ ID NO:2. Amino acid residues 28 to32 of SEQ ID NO:4 correspond to amino acid residues 27 to 32 of humanANP and are designated as SEQ ID NO:7.

FIG. 3 is a graph plotting plasma cGMP levels before and during BAA-NPinfusion (n=6).

FIG. 4 is a graph plotting urinary cGMP excretion before and duringBAA-NP infusion (n=6).

FIG. 5 is a graph plotting net renal generation of cGMP before andduring BAA-NP infusion (n=5).

FIG. 6 is a graph plotting urine flow before and during BAA-NP infusion(n=6).

FIG. 7 is a graph plotting urinary sodium excretion before and duringBAA-NP infusion (n=6).

FIG. 8 is a graph plotting glomerular filtration rate before and duringBAA-NP infusion (n=6).

FIG. 9 is a graph plotting renal blood flow before and during BAA-NPinfusion (n=6).

FIG. 10 is a graph plotting renal perfusion pressure before and duringBAA-NP infusion (n=6).

FIG. 11 is a graph plotting renal vascular resistance before and duringBAA-NP infusion (n=6).

FIG. 12 is a graph plotting proximal fractional reabsorption of sodiumbefore and during BAA-NP infusion (n=4).

FIG. 13 is a graph plotting distal fractional reabsorption of sodiumbefore and during BAA-NP infusion (n=4).

FIG. 14 is a graph plotting urinary potassium excretion before andduring BAA-NP infusion (n=6).

FIG. 15 is a graph plotting mean arterial pressure before and duringBAA-NP infusion (n=6).

FIG. 16 is a graph plotting pulmonary capillary wedge pressure beforeand during BAA-NP infusion (n=6).

FIG. 17 is a graph plotting right atrial pressure before and duringBAA-NP infusion (n=6).

FIG. 18 is a graph plotting pulmonary arterial pressure before andduring BAA-NP infusion (n=6).

FIG. 19 is a graph plotting cardiac output before and during BAA-NPinfusion (n=6).

FIG. 20 is a graph plotting systemic vascular resistance before andduring BAA-NP infusion (n=6).

FIG. 21 is a graph plotting pulmonary vascular resistance before andduring BAA-NP infusion (n=6).

FIG. 22 is a graph plotting hematocrit levels before and during BAA-NPinfusion (n=6).

FIG. 23 is a graph plotting plasma renin activity before and duringBAA-NP infusion (n=6).

FIG. 24 is a graph plotting plasma angiotensin ii before and duringBAA-NP infusion (n=6).

FIG. 25 is a graph plotting plasma aldosterone before and during BAA-NPinfusion (n=5).

FIG. 26 is a graph plotting plasma ANP immunoreactivity before andduring BAA-NP infusion (n=6).

FIG. 27 is a graph plotting urinary excretion of ANP before and duringBAA-NP infusion (n=6).

FIG. 28 is a graph plotting plasma CNP immunoreactivity before andduring BAA-NP infusion (n=6).

FIG. 29 is a graph plotting urinary CNP excretion before and duringBAA-NP infusion (n=5).

FIG. 30 is a graph plotting plasma cGMP before and during BUA-NPinfusion (n=6).

FIG. 31 is a graph plotting urinary cGMP excretion before and duringBUA-NP infusion (n=6).

FIG. 32 is a graph plotting net renal generation of cGMP before andduring BUA-NP infusion (n=6).

FIG. 33 is a graph plotting urine flow before and during BUA-NP infusion(n=6).

FIG. 34 is a graph plotting urinary sodium excretion before and duringBUA-NP infusion (n=6).

FIG. 35 is a graph plotting glomerular filtration rate before and duringBUA-NP infusion (n=6).

FIG. 36 is a graph plotting renal blood flow before and during BUA-NPinfusion (n=6).

FIG. 37 is a graph plotting renal perfusion pressure before and duringBUA-NP infusion (n=6).

FIG. 38 is a graph plotting renal vascular resistance before and duringBUA-NP infusion (n=6).

FIG. 39 is a graph plotting proximal fractional reabsorption of sodiumbefore and during BUA-NP infusion (n=4).

FIG. 40 is a graph plotting distal fractional reabsorption of sodiumbefore and during BUA-NP infusion (n=4).

FIG. 41 is a graph plotting urinary potassium excretion before andduring BUA-NP infusion (n=6).

FIG. 42 is a graph plotting mean arterial pressure before and duringBUA-NP infusion (n=6).

FIG. 43 is a graph plotting pulmonary capillary wedge pressure beforeand during BUA-NP infusion (n=6).

FIG. 44 is a graph plotting right atrial pressure before and duringBUA-NP infusion (n=6).

FIG. 45 is a graph plotting pulmonary arterial pressure before andduring BUA-NP infusion (n=6).

FIG. 46 is a graph plotting cardiac output before and during BUA-NPinfusion (n=6).

FIG. 47 is a graph plotting systemic vascular resistance before andduring BUA-NP infusion (n=6).

FIG. 48 is a graph plotting pulmonary vascular resistance before andduring BUA-NP infusion (n=6).

FIG. 49 is a graph plotting hematocrit levels before and during BUA-NPinfusion (n=6).

FIG. 50 is a graph plotting plasma renin activity before and duringBUA-NP infusion (n=5).

FIG. 51 is a graph plotting plasma angiotensin II before and duringBUA-NP infusion (n=5).

FIG. 52 is a graph plotting plasma aldosterone before and during BUA-NPinfusion (n=3).

FIG. 53 is a graph plotting plasma ANP immunoreactivity before andduring BUA-NP infusion (n=6).

FIG. 54 is a graph plotting urinary excretion of ANP before and duringBUA-NP infusion (n=6).

FIG. 55 is a graph plotting plasma CNP immunoreactivity before andduring BUA-NP infusion (n=6).

FIG. 56 is a graph plotting urinary CNP excretion before and duringBUA-NP infusion (n=6).

FIG. 57 is a graph plotting mean arterial pressure for the indicatedpolypeptides. The comparison was performed using an equimolar dose ofhuman BNP.

FIG. 58 is a graph plotting renal perfusion pressure for the indicatedpolypeptides. The comparison was performed using an equimolar dose ofhuman BNP.

FIG. 59 is a graph plotting the change in renal perfusion pressure frombaseline at 30 minutes of infusion with the indicated peptide.

FIG. 60 is a graph plotting the change in renal perfusion pressure frombaseline at 30 minutes of infusion with the indicated peptide.

FIG. 61 is a graph plotting the change in mean arterial pressure frombaseline at 30 minutes of infusion with the indicated peptide.

FIG. 62 is a graph plotting the change in mean arterial pressure frombaseline at 60 minutes of infusion with the indicated peptide.

FIG. 63 is a graph plotting plasma cGMP levels (pmol/mL) before andafter (30 minutes and 60 minutes) infusion with the indicated peptides.

FIG. 64 is a graph plotting urinary cGMP excretion (pmol/min) before andafter (30 minutes and 60 minutes) infusion with the indicated peptides.

FIG. 65 is a graph plotting net renal cGMP generation (pmol/min) beforeand after (30 minutes and 60 minutes) infusion with the indicatedpeptides.

FIG. 66 is a graph plotting urinary sodium excretion (μEq/min) beforeand after (30 minutes and 60 minutes) infusion with the indicatedpeptides.

FIG. 67 is a graph plotting urine flow (mL/min) before and after (30minutes and 60 minutes) infusion with the indicated peptides.

FIG. 68 is a graph plotting glomerular filtration rate (mL/min) beforeand after (30 minutes and 60 minutes) infusion with the indicatedpeptides.

FIG. 69 is a graph plotting mean arterial pressure (mmHg) before andafter (30 minutes and 60 minutes) infusion with the indicated peptides.

FIG. 70 is a graph plotting the proximal fractional reabsorption ofsodium (%) before and after (30 minutes and 60 minutes) infusion withthe indicated peptides.

FIG. 71 is a graph plotting the distal fractional reabsorption of sodium(%) before and after (30 minutes and 60 minutes) infusion with theindicated peptides.

FIG. 72 is a graph plotting pulmonary capillary pressure (mmHg) beforeand after (30 minutes and 60 minutes) infusion with the indicatedpeptides.

FIG. 73 is a graph plotting right arterial pressure (mmHg) before andafter (30 minutes and 60 minutes) infusion with the indicated peptides.

DETAILED DESCRIPTION

This document relates to natriuretic polypeptides. For example, thisdocument provides methods and materials related to natriureticpolypeptides and the use of natriuretic polypeptides to treatcardiovascular conditions (e.g., acute decompensated heart failure,acute coronary syndromes, and ventricular remodeling post-myocardialinfarction) and renal conditions (e.g., perioperative renal dysfunction,renal dysfunction secondary to heart failure, and diabetic nephropathy).

A polypeptide provided herein can have any sequence and can have anylength. For example, a polypeptide provided herein can include thesequence set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In somecases, a polypeptide provided herein can contain an amino acid sequencethat aligns to (a) the sequence set forth in SEQ ID NO:1 with three orless (e.g., two or less, one, or zero) amino acid additions, deletions,substitutions, or combinations thereof, (b) the sequence set forth inSEQ ID NO:2 with five or less (e.g., four or less, three or less, two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof and (c) the sequence set forth in SEQ ID NO:3 withthree or less (e.g., two or less, one, or zero) amino acid additions,deletions, substitutions, or combinations thereof. For example, apolypeptide provided herein can contain the sequence set forth in SEQ IDNO:1 with the exception that the first serine residue or the last serineresidue of SEQ ID NO:1 is deleted or replaced with a different aminoacid residue.

In some cases, a polypeptide provided herein can include the sequenceset forth in SEQ ID NO:6, SEQ ID NO:2, and SEQ ID NO:7. In some cases, apolypeptide provided herein can contain an amino acid sequence thataligns to (a) the sequence set forth in SEQ ID NO:6 with three or less(e.g., two or less, one, or zero) amino acid additions, deletions,substitutions, or combinations thereof, (b) the sequence set forth inSEQ ID NO:2 with five or less (e.g., four or less, three or less, two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof and (c) the sequence set forth in SEQ ID NO:7 withthree or less (e.g., two or less, one, or zero) amino acid additions,deletions, substitutions, or combinations thereof. For example, apolypeptide provided herein can contain the sequence set forth in SEQ IDNO:6 with the exception that the first occurring proline residue or thelast occurring serine residue of SEQ ID NO:6 is deleted or replaced witha different amino acid residue. In some cases, a polypeptide providedherein can contain an amino acid sequence that aligns to (a) thesequence set forth in SEQ ID NO:6 with three or less (e.g., two or less,one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof, provided that the polypeptide contains the TAPRamino acid sequence, (b) the sequence set forth in SEQ ID NO:2 with fiveor less (e.g., four or less, three or less, two or less, one, or zero)amino acid additions, deletions, substitutions, or combinations thereofand (c) the sequence set forth in SEQ ID NO:7 with three or less (e.g.,two or less, one, or zero) amino acid additions, deletions,substitutions, or combinations thereof. The presence of an N-terminalTAPR amino acid sequence can enhance the cGMP-activating and renalactions of the polypeptide.

Amino acid substitutions can be conservative amino acid substitutions.Conservative amino acid substitutions can be, for example,aspartic-glutamic as acidic amino acids; lysine/arginine/histidine asbasic amino acids; leucine/isoleucine, methionine/valine, alanine/valineas hydrophobic amino acids; serine/glycine/alanine/threonine ashydrophilic amino acids. Conservative amino acid substitutions alsoinclude groupings based on side chains. For example, a group of aminoacids having aliphatic side chains is glycine, alanine, valine, leucine,and isoleucine; a group of amino acids having aliphatic-hydroxyl sidechains is serine and threonine; a group of amino acids havingamide-containing side chains is asparagine and glutamine; a group ofamino acids having aromatic side chains is phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains is lysine,arginine, and histidine; and a group of amino acids havingsulfur-containing side chains is cysteine and methionine. After makingan amino acid substitution, the activities of the polypeptide containingthe amino acid substitution can be assessed using the assays describedherein.

In some cases, a polypeptide provided herein can contain (a) a firstamino acid sequence that either is set forth in SEQ ID NO:1 or aligns tothe sequence set forth in SEQ ID NO:1 with three or less (e.g., two orless, one, or zero) amino acid deletions, substitutions, or combinationsthereof, (b) a second amino acid sequence that either is set forth inSEQ ID NO:2 or aligns to the sequence set forth in SEQ ID NO:2 with fiveor less (e.g., four or less, three or less, two or less, one, or zero)amino acid additions, substitutions, or combinations thereof, and (c) athird amino acid sequence that either is set forth in SEQ ID NO:3 oraligns to the sequence set forth in SEQ ID NO:3 with three or less(e.g., two or less, one, or zero) amino acid deletions, substitutions,or combinations thereof. For example, a polypeptide provided herein cancomprise or consist of the sequence set forth in SEQ ID NO:4.

In some cases, a polypeptide provided herein can contain (a) a firstamino acid sequence that either is set forth in SEQ ID NO:6 or aligns tothe sequence set forth in SEQ ID NO:6 with three or less (e.g., two orless, one, or zero) amino acid deletions, substitutions, or combinationsthereof, (b) a second amino acid sequence that either is set forth inSEQ ID NO:2 or aligns to the sequence set forth in SEQ ID NO:2 with fiveor less (e.g., four or less, three or less, two or less, one, or zero)amino acid additions, substitutions, or combinations thereof, and (c) athird amino acid sequence that either is set forth in SEQ ID NO:7 oraligns to the sequence set forth in SEQ ID NO:7 with three or less(e.g., two or less, one, or zero) amino acid deletions, substitutions,or combinations thereof. For example, a polypeptide provided herein cancomprise or consist of the sequence set forth in SEQ ID NO:5.

A polypeptide provided herein can have any length. For example, apolypeptide provided herein can be between 23 and 45 (e.g., between 25and 45, between 26 and 44, between 27 and 43, between 28 and 42, between29 and 41, between 30 and 40, between 31 and 39, between 23 and 35,between 25 and 30, or between 30 and 35) amino acid residues in length.It will be appreciated that a polypeptide with a length of 25 or 45amino acid residues is a polypeptide with a length between 25 and 45amino acid residues.

In some cases, a polypeptide provided herein can be a substantially purepolypeptide. As used herein, the term “substantially pure” withreference to a polypeptide means that the polypeptide is substantiallyfree of other polypeptides, lipids, carbohydrates, and nucleic acid withwhich it is naturally associated. Thus, a substantially pure polypeptideis any polypeptide that is removed from its natural environment and isat least 60 percent pure or is any chemically synthesized polypeptide. Asubstantially pure polypeptide can be at least about 60, 65, 70, 75, 80,85, 90, 95, or 99 percent pure. Typically, a substantially purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel.

A polypeptide provide herein can be obtained by expression of arecombinant nucleic acid encoding the polypeptide or by chemicalsynthesis (e.g., using solid phase polypeptide synthesis methods or anpeptide synthesizer such as an ABI 431A Peptide Synthesizer; AppliedBiosystems; Foster City, Calif.). For example, standard recombinanttechnology using expression vectors encoding a polypeptide provideherein can be used. The resulting polypeptides then can be purifiedusing, for example, affinity chromatographic techniques and HPLC. Theextent of purification can be measured by any appropriate method,including but not limited to: column chromatography, polyacrylamide gelelectrophoresis, or high-performance liquid chromatography. Apolypeptide provide herein can be designed or engineered to contain atag sequence that allows the polypeptide to be purified (e.g., capturedonto an affinity matrix). For example, a tag such as c-myc,hemagglutinin, polyhistidine, or Flag™ tag (Kodak) can be used to aidpolypeptide purification. Such tags can be inserted anywhere within thepolypeptide including at either the carboxyl or amino termini. Otherfusions that can be used include enzymes that aid in the detection ofthe polypeptide, such as alkaline phosphatase.

A polypeptide provided herein can be produced to contain three regions,a first region that includes an N-terminus (e.g., an N-terminus sequencefrom a human ANP or urodilatin polypeptide), a second region thatincludes a ring structure of a mature natriuretic polypeptide such as ahuman BNP polypeptide, and third region that includes a C-terminus(e.g., the C-terminus sequence from a human ANP or urodilatinpolypeptide). For example, a polypeptide provided herein can be producedto contain a first region that includes an N-terminus sequence from ahuman ANP or urodilatin polypeptide, a second region that includes aring structure of a mature natriuretic polypeptide such as a human BNPpolypeptide, and third region that includes the C-terminus sequence froma human ANP or urodilatin polypeptide.

A polypeptide provided herein can be used to treat cardiovasculardiseases, congestive heart failure, myocardial infarction, coronaryartery diseases, renal diseases, hepatic diseases, cancer, metabolicdiseases, or combinations thereof. For example, a BAA-NP polypeptidehaving the amino acid sequence set forth in SEQ ID NO:4 or a BUA-NPpolypeptide having the amino acid sequence set forth in SEQ ID NO:5 canbe administered to a human having coronary artery disease underconditions wherein the severity of the human's coronary artery diseasesymptoms is reduced.

A polypeptide provided herein can be formulated as a pharmaceuticalcomposition by admixture with pharmaceutically acceptable non-toxicexcipients or carriers. Such compositions can be administered to asubject in need thereof in an amount effective to treat, for example,heart, liver, kidney, or other sodium retaining conditions.Pharmaceutical compositions can be prepared for parenteraladministration, particularly in the form of liquid solutions orsuspensions in aqueous physiological buffer solutions; for oraladministration, particularly in the form of tablets or capsules; or forintranasal administration, particularly in the form of powders, nasaldrops, or aerosols. Compositions for other routes of administration canbe prepared as desired using appropriate methods.

Formulations for parenteral administration can include as commonexcipients, sterile water, saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenatednaphthalenes, and combinations thereof. In some cases, biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer,polyoxethylene-polyoxypropylene copolymers, or combinations thereof canbe used as excipients for controlling the release of the polypeptide invivo. Other suitable parenteral delivery systems that can be usedinclude, without limitation, ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, liposomes, and combinationsthereof. Formulations for inhalation administration can includeexcipients such as lactose. Inhalation formulations can be aqueoussolutions containing, for example, polyoxyethylene-9-lauryl ether,glycocholate, deoxycholate, or combinations thereof, or they can be oilysolutions for administration in the form of nasal drops. If desired, acomposition containing a polypeptide provided herein can be formulatedas gel to be applied intranasally. Formulations for parenteraladministration can include glycocholate for buccal administration.

For oral administration, tablets or capsules can be prepared usingappropriate methods with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone,or hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g. magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). Tablets can be coated using appropriate methods.Preparations for oral administration can be formulated to givecontrolled release of the polypeptide.

Nasal preparations can be presented in a liquid form or as a dryproduct. Nebulised aqueous suspensions or solutions can include carriersor excipients to adjust pH and/or tonicity.

Nucleic Acids Encoding Polypeptides

This document also provides isolated nucleic acids that encode one ormore of the polypeptides provided herein. The term “isolated” as usedherein with reference to nucleic acid refers to a naturally-occurringnucleic acid that is not immediately contiguous with both of thesequences with which it is immediately contiguous (one on the 5′ end andone on the 3′ end) in the naturally-occurring genome of the organismfrom which it is derived. For example, an isolated nucleic acid can be,without limitation, a recombinant DNA molecule of any length, providedone of the nucleic acid sequences normally found immediately flankingthat recombinant DNA molecule in a naturally-occurring genome is removedor absent. Thus, an isolated nucleic acid includes, without limitation,a recombinant DNA that exists as a separate molecule (e.g., a cDNA or agenomic DNA fragment produced by PCR or restriction endonucleasetreatment) independent of other sequences as well as recombinant DNAthat is incorporated into a vector, an autonomously replicating plasmid,a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into thegenomic DNA of a prokaryote or eukaryote. In addition, an isolatednucleic acid can include a recombinant DNA molecule that is part of ahybrid or fusion nucleic acid sequence.

The term “isolated” as used herein with reference to nucleic acid alsoincludes any non-naturally-occurring nucleic acid sincenon-naturally-occurring nucleic acid sequences are not found in natureand do not have immediately contiguous sequences in anaturally-occurring genome. For example, non-naturally-occurring nucleicacid such as an engineered nucleic acid is considered to be isolatednucleic acid. Engineered nucleic acid (e.g., a nucleic acid encoding apolypeptide comprising or consisting of the amino acid sequence setforth in SEQ ID NO:4 or SEQ ID NO:5) can be made using common molecularcloning or chemical nucleic acid synthesis techniques. Isolatednon-naturally-occurring nucleic acid can be independent of othersequences, or incorporated into a vector, an autonomously replicatingplasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), orthe genomic DNA of a prokaryote or eukaryote. In addition, anon-naturally-occurring nucleic acid can include a nucleic acid moleculethat is part of a hybrid or fusion nucleic acid sequence. A nucleic acidexisting among hundreds to millions of other nucleic acids within, forexample, cDNA libraries or genomic libraries, or gel slices containing agenomic DNA restriction digest, is not to be considered an isolatednucleic acid.

As used herein, the term “nucleic acid” refers to both RNA and DNA,including mRNA, cDNA, genomic DNA, synthetic (e.g., chemicallysynthesized) DNA, and nucleic acid analogs. The nucleic acid can bedouble-stranded or single-stranded, and where single-stranded, can bethe sense strand or the antisense strand. In addition, nucleic acid canbe circular or linear. Nucleic acid analogs can be modified at the basemoiety, sugar moiety, or phosphate backbone to improve, for example,stability, hybridization, or solubility of a nucleic acid. Modificationsat the base moiety include deoxyuridine for deoxythymidine, and5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidine fordeoxycytidine. Modifications of the sugar moiety can includemodification of the 2′ hydroxyl of the ribose sugar to form 2′-O-methylor 2′-O-allyl sugars. The deoxyribose phosphate backbone can be modifiedto produce morpholino nucleic acids, in which each base moiety is linkedto a six-membered, morpholino ring, or peptide nucleic acids, in whichthe deoxyphosphate backbone is replaced by a pseudopeptide backbone andthe four bases are retained. See, for example, Summerton and WellerAntisense Nucleic Acid Drug Dev., 7:187-195 (1997); and Hyrup et al.Bioorgan. Med. Chem., 4:5-23 (1996). In addition, the deoxyphosphatebackbone can be replaced with, for example, a phosphorothioate orphosphorodithioate backbone, a phosphoroamidite, or an alkylphosphotriester backbone.

A nucleic acid provided herein can comprise or consist of a sequencethat encodes the amino acid sequence set forth in SEQ ID NO:4 or SEQ IDNO:5. For example, such a nucleic acid can contain the human nucleicacid sequence for BNP and ANP engineered to encode the amino acidsequence set forth in SEQ ID NO:4. In some cases, such a nucleic acidcan contain the human nucleic acid sequence for BNP, urodilatin, and ANPengineered to encode the amino acid sequence set forth in SEQ ID NO:5.

Typically, an isolated nucleic acid provided herein is at least 10nucleotides in length (e.g., 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,200, 300, 350, 400, or more nucleotides in length). Nucleic acidmolecules that are less than full-length can be useful, for example, asprimers or probes for diagnostic purposes. Isolated nucleic acidmolecules can be produced by standard techniques, including, withoutlimitation, common molecular cloning and chemical nucleic acid synthesistechniques. For example, polymerase chain reaction (PCR) techniques canbe used. PCR refers to a procedure or technique in which target nucleicacids are enzymatically amplified. Sequence information from the ends ofthe region of interest or beyond typically is employed to designoligonucleotide primers that are identical in sequence to oppositestrands of the template to be amplified. PCR can be used to amplifyspecific sequences from DNA as well as RNA, including sequences fromtotal genomic DNA or total cellular RNA. Primers typically are 15 to 50nucleotides in length, but can range from 10 nucleotides to hundreds ofnucleotides in length. For example, a primer can be 12, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, or 45nucleotides in length. A primer can be purified from a restrictiondigest by conventional methods, or can be chemically synthesized.Primers typically are single-stranded for maximum efficiency inamplification, but a primer can be double-stranded. Double-strandedprimers are first denatured (e.g., treated with heat) to separate thestrands before use in amplification. General PCR techniques aredescribed, for example in PCR Primer: A Laboratory Manual, ed. byDieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.When using RNA as a source of template, reverse transcriptase can beused to synthesize a complementary DNA (cDNA) strand. Ligase chainreaction, strand displacement amplification, self-sustained sequencereplication or nucleic acid sequence-based amplification also can beused to obtain isolated nucleic acids as described elsewhere (Lewis,Genetic Engineering News, 12(9):1 (1992); Guatelli et al., Proc. Natl.Acad. Sci. USA, 87:1874-1878 (1990); and Weiss, Science, 254:1292(1991)).

Isolated nucleic acids also can be chemically synthesized, either as asingle nucleic acid molecule (e.g., using automated DNA synthesis in the3′ to 5′ direction using phosphoramidite technology) or as a series ofoligonucleotides. For example, one or more pairs of longoligonucleotides (e.g., >100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a single, double-strandednucleic acid molecule per oligonucleotide pair, which then can beligated into a vector.

Isolated nucleic acids also can be obtained by mutagenesis. For example,a nucleic acid sequence encoding a polypeptide having the sequence setforth in SEQ ID NO:1, 2, 3, 4, 5, 6, or 7 can be mutated using standardtechniques such as, for example, oligonucleotide-directed mutagenesisand/or site-directed mutagenesis through PCR. See, Short Protocols inMolecular Biology, Chapter 8, Green Publishing Associates and John Wiley& Sons, Edited by Ausubel et al., 1992. Such mutations includeadditions, deletions, substitutions, and combinations thereof.

Vectors and Host Cells

This document also provides vectors containing a nucleic acid providedherein. As used herein, a “vector” is a replicon, such as a plasmid,phage, or cosmid, into which another DNA segment can be inserted so asto bring about the replication of the inserted segment. A vector can bean expression vector. An “expression vector” is a vector that includesone or more expression control sequences, and an “expression controlsequence” is a DNA sequence that controls and regulates thetranscription and/or translation of another DNA sequence.

In an expression vector provided herein, the nucleic acid can beoperably linked to one or more expression control sequences. As usedherein, “operably linked” means incorporated into a genetic construct sothat expression control sequences effectively control expression of acoding sequence of interest. Examples of expression control sequencesinclude promoters, enhancers, and transcription terminating regions. Apromoter is an expression control sequence composed of a region of a DNAmolecule, typically within 100 nucleotides upstream of the point atwhich transcription starts (generally near the initiation site for RNApolymerase II). To bring a coding sequence under the control of apromoter, it can be necessary to position the translation initiationsite of the translational reading frame of the polypeptide between oneand about fifty nucleotides downstream of the promoter. Enhancersprovide expression specificity in terms of time, location, and level.Unlike promoters, enhancers can function when located at variousdistances from the transcription site. An enhancer also can be locateddownstream from the transcription initiation site. A coding sequence is“operably linked” and “under the control” of expression controlsequences in a cell when RNA polymerase is able to transcribe the codingsequence into mRNA, which then can be translated into the polypeptideencoded by the coding sequence.

Suitable expression vectors include, without limitation, plasmids andviral vectors derived from, for example, bacteriophage, baculoviruses,tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses,poxviruses, adenoviruses, and adeno-associated viruses. Numerous vectorsand expression systems are commercially available from such corporationsas Novagen (Madison, Wis.), Clontech Laboratories (Mountain View,Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies(Carlsbad, Calif.).

An expression vector can include a tag sequence designed to facilitatesubsequent manipulation of the expressed nucleic acid sequence (e.g.,purification or localization). Tag sequences, such as green fluorescentprotein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc,hemagglutinin, or Flag™ tag (Kodak, New Haven, Conn.) sequencestypically are expressed as a fusion with the encoded polypeptide. Suchtags can be inserted anywhere within the polypeptide including at eitherthe carboxyl or amino terminus.

This document also provides host cells containing a nucleic acidmolecule and/or nucleic acid vector provided herein. The term “hostcell” refers to prokaryotic cells and eukaryotic cells into which anucleic acid molecule or vector can be introduced. Any method can beused to introduce nucleic acid into a cell. For example, calciumphosphate precipitation, electroporation, heat shock, lipofection,microinjection, and viral-mediated nucleic acid transfer can be usedintroduce nucleic acid into cells. In addition, naked DNA can bedelivered directly to cells in vivo as described elsewhere (U.S. Pat.Nos. 5,580,859 and 5,589,466).

Detecting Polypeptides

This document provides methods and materials for detecting a polypeptideprovided herein. Such methods and materials can be used to monitorpolypeptide levels within a mammal receiving the polypeptide as atherapeutic. A polypeptide provided herein (e.g., a BAA-NP polypeptidehaving the amino acid sequence set forth in SEQ ID NO:4 or a BUA-NPpolypeptide having the amino acid sequence set forth in SEQ ID NO:5) canbe detected, for example, immunologically using one or more antibodies.As used herein, the term “antibody” includes intact molecules as well asfragments thereof that are capable of binding to an epitopic determinantof a polypeptide provided herein. The term “epitope” refers to anantigenic determinant on an antigen to which the paratope of an antibodybinds. Epitopic determinants usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chains,and typically have specific three-dimensional structuralcharacteristics, as well as specific charge characteristics. Epitopesgenerally have at least five contiguous amino acids (a continuousepitope), or alternatively can be a set of noncontiguous amino acidsthat define a particular structure (e.g., a conformational epitope). Theterm “antibody” includes polyclonal antibodies, monoclonal antibodies,humanized or chimeric antibodies, single chain Fv antibody fragments,Fab fragments, and F(ab)2 fragments. Polyclonal antibodies areheterogenous populations of antibody molecules that are contained in thesera of the immunized animals. Monoclonal antibodies are homogeneouspopulations of antibodies to a particular epitope of an antigen.

Antibody fragments that have specific binding affinity for a polypeptideprovided herein (e.g., a BAA-NP polypeptide having the amino acidsequence set forth in SEQ ID NO:4 or a BUA-NP polypeptide having theamino acid sequence set forth in SEQ ID NO:5) can be generated by knowntechniques. For example, F(ab′)2 fragments can be produced by pepsindigestion of the antibody molecule; Fab fragments can be generated byreducing the disulfide bridges of F(ab′)2 fragments. In some cases, Fabexpression libraries can be constructed. See, for example, Huse et al.,Science, 246:1275 (1989). Once produced, antibodies or fragments thereofcan be tested for recognition of a polypeptide provided herein bystandard immunoassay methods including ELISA techniques,radioimmunoassays, and Western blotting. See, Short Protocols inMolecular Biology, Chapter 11, Green Publishing Associates and JohnWiley & Sons, Edited by Ausubel, F. M et al., 1992.

In immunological assays, an antibody having specific binding affinityfor a polypeptide provided herein or a secondary antibody that binds tosuch an antibody can be labeled, either directly or indirectly. Suitablelabels include, without limitation, radionuclides (e.g., ¹²⁵I, ¹³¹I,³⁵S, ³H, ³²P, ³³P, or ¹⁴C), fluorescent moieties (e.g., fluorescein,FITC, PerCP, rhodamine, or PE), luminescent moieties (e.g., Qdot™nanoparticles supplied by Invitrogen (Carlsbad, Calif.)), compounds thatabsorb light of a defined wavelength, or enzymes (e.g., alkalinephosphatase or horseradish peroxidase). Antibodies can be indirectlylabeled by conjugation with biotin then detected with avidin orstreptavidin labeled with a molecule described above. Methods ofdetecting or quantifying a label depend on the nature of the label andare known in the art. Examples of detectors include, without limitation,x-ray film, radioactivity counters, scintillation counters,spectrophotometers, colorimeters, fluorometers, luminometers, anddensitometers. Combinations of these approaches (including “multi-layer”assays) familiar to those in the art can be used to enhance thesensitivity of assays.

Immunological assays for detecting a polypeptide provided herein can beperformed in a variety of known formats, including sandwich assays,competition assays (competitive RIA), or bridge immunoassays. See, forexample, U.S. Pat. Nos. 5,296,347; 4,233,402; 4,098,876; and 4,034,074.Methods of detecting a polypeptide provided herein generally includecontacting a biological sample with an antibody that binds to apolypeptide provided herein and detecting binding of the polypeptide tothe antibody. For example, an antibody having specific binding affinityfor a polypeptide provided herein can be immobilized on a solidsubstrate by any of a variety of methods known in the art and thenexposed to the biological sample. Binding of the polypeptide to theantibody on the solid substrate can be detected by exploiting thephenomenon of surface plasmon resonance, which results in a change inthe intensity of surface plasmon resonance upon binding that can bedetected qualitatively or quantitatively by an appropriate instrument,e.g., a Biacore apparatus (Biacore International AB, Rapsgatan, Sweden).In some cases, the antibody can be labeled and detected as describedabove. A standard curve using known quantities of a polypeptide providedherein can be generated to aid in the quantitation of the levels of thepolypeptide.

In some embodiments, a “sandwich” assay in which a capture antibody isimmobilized on a solid substrate can be used to detect the presence,absence, or level of a polypeptide provided herein. The solid substratecan be contacted with the biological sample such that any polypeptide ofinterest in the sample can bind to the immobilized antibody. Thepresence, absence, or level of the polypeptide bound to the antibody canbe determined using a “detection” antibody having specific bindingaffinity for the polypeptide. In some embodiments, a capture antibodycan be used that has binding affinity for ANP, BNP, or urodilatin aswell as a polypeptide provided herein. In this embodiment, a detectionantibody can be used that has specific binding affinity for a particularpolypeptide provided herein (e.g., a BAA-NP polypeptide having the aminoacid sequence set forth in SEQ ID NO:4 or a BUA-NP polypeptide havingthe amino acid sequence set forth in SEQ ID NO:5). It is understood thatin sandwich assays, the capture antibody should not bind to the sameepitope (or range of epitopes in the case of a polyclonal antibody) asthe detection antibody. Thus, if a monoclonal antibody is used as acapture antibody, the detection antibody can be another monoclonalantibody that binds to an epitope that is either physically separatedfrom or only partially overlaps with the epitope to which the capturemonoclonal antibody binds, or a polyclonal antibody that binds toepitopes other than or in addition to that to which the capturemonoclonal antibody binds. If a polyclonal antibody is used as a captureantibody, the detection antibody can be either a monoclonal antibodythat binds to an epitope that is either physically separated from orpartially overlaps with any of the epitopes to which the capturepolyclonal antibody binds, or a polyclonal antibody that binds toepitopes other than or in addition to that to which the capturepolyclonal antibody binds. Sandwich assays can be performed as sandwichELISA assays, sandwich Western blotting assays, or sandwichimmunomagnetic detection assays.

Suitable solid substrates to which an antibody (e.g., a captureantibody) can be bound include, without limitation, microtiter plates,tubes, membranes such as nylon or nitrocellulose membranes, and beads orparticles (e.g., agarose, cellulose, glass, polystyrene, polyacrylamide,magnetic, or magnetizable beads or particles). Magnetic or magnetizableparticles can be particularly useful when an automated immunoassaysystem is used.

Antibodies having specific binding affinity for a polypeptide providedherein can be produced through standard methods. For example, apolypeptide can be recombinantly produced as described above, can bepurified from a biological sample (e.g., a heterologous expressionsystem), or can be chemically synthesized, and used to immunize hostanimals, including rabbits, chickens, mice, guinea pigs, or rats. Forexample, a polypeptide having the amino acid sequence set forth in SEQID NO:4 or SEQ ID NO:5, or fragments thereof that are at least six aminoacids in length, can be used to immunize an animal Various adjuvantsthat can be used to increase the immunological response depend on thehost species and include Freund's adjuvant (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin and dinitrophenol. Monoclonal antibodies canbe prepared using a polypeptide provided herein and standard hybridomatechnology. In particular, monoclonal antibodies can be obtained by anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture such as described by Kohler et al.,Nature, 256:495 (1975), the human B-cell hybridoma technique (Kosbor etal., Immunology Today, 4:72 (1983); Cole et al., Proc. Natl. Acad. Sci.USA, 80:2026 (1983)), and the EBV-hybridoma technique (Cole et al.,“Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, Inc., pp.77-96 (1983)). Such antibodies can be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, IgD, and any subclass thereof. Thehybridoma producing the monoclonal antibodies can be cultivated in vitroand in vivo.

Other techniques for detecting a polypeptide provided herein includemass-spectrophotometric techniques such as electrospray ionization(ESI), and matrix-assisted laser desorption-ionization (MALDI). See, forexample, Gevaert et al., Electrophoresis, 22(9):1645-51 (2001); Chaurandet al., J. Am. Soc. Mass Spectrom., 10(2):91-103 (1999). Massspectrometers useful for such applications are available from AppliedBiosystems (Foster City, Calif.); Bruker Daltronics (Billerica, Mass.);and Amersham Pharmacia (Sunnyvale, Calif.).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES

A polypeptide with the sequence set forth in FIG. 1 was designed andsynthesized using an ABI 431A Peptide Synthesizer. This polypeptide isreferred to as a BAA-NP polypeptide (FIG. 1). The synthesized BAA-NPpolypeptide was confirmed by high-performance liquid chromatography andmass spectrometry. Its molecular weight was 3158.60, and its amino acidsequence is SLRRSSCFGRKMDRISSSSG-LGCNSFRY (SEQ ID NO:4) with a disulfidebridge joining the Cys residues.

A polypeptide with the sequence set forth in FIG. 2 was designed andsynthesized using an ABI 431A Peptide Synthesizer. This polypeptide isreferred to as a BUA-NP polypeptide (FIG. 2). The synthesized BUA-NPpolypeptide was confirmed by high-performance liquid chromatography andmass spectrometry. Its molecular weight was 3582.08, and its amino acidsequence is TAPRSLRRSSCFGRKMDRIS-SSSGLGCNSFRY (SEQ ID NO:5) with adisulfide bridge joining the Cys residues.

BAA-NP and BUA-NP were reconstituted in normal saline and were infusedat 1 mL/minute. Dogs were maintained on a Na⁺-controlled diet (Hill'si/d® canine diet, Hill's Pet Nutrition, Inc., Topeka, Kans.). In theevening before the experiment, the dog was fasted with ad lib access towater and was given lithium carbonate 300 mg orally for assessment ofrenal tubular function on the next day.

On the day of the experiment, the dog was anesthetized withpentobarbital sodium (induction 6-20 mg/kg i.v., maintenance 5-15mg/kg/h i.v.) and fentanyl (0.04-0.12 mg/kg i.v., maintenance 0.04-0.18mg/kg/h), and was intubated and mechanically ventilated (HarvardApparatus, Holliston, Mass.) with 3 L/minute of 02 (tidal volume 15mL/kg, 12 cycles/minute). The femoral artery was cannulated for bloodpressure monitoring and for blood sampling. The femoral vein wascannulated for infusion of inulin and normal saline. The saphenous veinwas cannulated for polypeptide infusion. A balloon-tipped thermodilutioncatheter (Edwards Lifesciences, Irvine, Calif.) was used for monitoringhemodynamics and body temperature (maintained at 38±1° C.). Cardiacoutput (CO) was measured in triplicate and averaged (model 9510-A,American Edwards Laboratories, Irvine, Calif.). Hemodynamic data weredigitally recorded and analyzed (Sonometrics Corporation, London, ON).The left kidney was exposed via a flank incision. The ureter wascannulated for timed urine collection. An electromagnetic flow probe wasplaced on the renal artery for measuring renal blood flow (Burnett etal., Am. J. Physiol., 247(5 Pt 2):F863-6 (1984)). A weight-adjustedbolus of inulin was given, followed by an inulin infusion (1 mL/minute)to achieve plasma levels of 40-60 mg/dL (Burnett et al., Am. J.Physiol., 247(5 Pt 2):F863-6 (1984); Chen et al., Am. J. Physiol. Regul.Integr. Comp. Physiol., 288(5):R1093-7 (2005); and Margulies et al., J.Clin. Invest., 88(5):1636-42 (1991)), for measuring GFR by inulinclearance. Normal saline was infused (1 mL/minute) and was temporarilydiscontinued during peptide infusion (1 mL/minute).

Following equilibration of approximately 60 minutes in duration, theBAA-NP or BUA-NP polypeptide was infused for 75 minutes. Hemodynamic andrenal data, as well as blood and urine samples, were collected for each30-minute clearance: pre-infusion, at 30 and 60 minutes of infusion(time denotes mid-clearance). Inulin clearance was used for measuringglomerular filtration rate (GFR). The lithium clearance technique wasused for quantifying proximal and distal fractional reabsorption of Na⁺(PFRNa and DFRNa, respectively).

Plasma and urinary ANP, CNP, and cGMP were measured by radioimmunoassays(RIA) (Chen et al., Am. J. Physiol. Regul. Integr. Comp. Physiol.,288(5):R1093-7 (2005); Burnett et al., Science, 231(4742):1145-7 (1986);Steiner et al., J. Biol. Chem., 247(4):1106-13 (1972); Supaporn et al.,Kidney Int., 50(5):1718-25 (1996); and Cataliotti et al., Am. J.Physiol., 283(3):F464-72 (2002)). Plasma renin activity (Haber et al.,J. Clin. Endocrinol. Metab., 29(10):1349-55 (1969)), angiotensin II(Luchner et al., Hypertension, 28(3):472-7 (1996)), and aldosterone(Sancho and Haber, J. Clin. Endocrinol. Metab., 47(2):391-6 (1978)) werequantified. Plasma and urinary lithium levels were measured by flameemission spectrophotometry (model 357, Instrumentation Laboratory,Wilmington, Mass.) (Margulies et al., J. Clin. Invest., 88(5):1636-42(1991)). The lithium clearance technique (CLLi) was used to assess PFRNaand DFRNa as follows (Chen et al., Am. J. Physiol. Regal. Integr. Comp.Physiol., 288(5):R1093-7 (2005)): PFRNa=[1−(CLLi/GFR)]×100 andDFRNa=[(CLLi−CLNa)/CLLi]×100, where CLLi=[urine Li⁺]×urine flow/[plasmaLi⁺] and CLNa=[urine Na⁺]×urine flow/[plasma Na]. Net renal productionof cGMP was determined (Margulies et al., J. Clin. Invest.,88(5):1636-42 (1991)): (urinary cGMP×urine flow rate)−(plasma cGMP×GFR).

In addition, comparisons were made with an equimolar dose of human BNP(n=7) for mean arterial pressure and renal perfusion pressure (thelatter was calculated by mean arterial pressure−right atrial pressure).

Comparisons of the data at 30 and 60 minutes of infusion vs.pre-infusion were made by one-way repeated measures ANOVA followed byDunnett's multiple comparison test. Between-group comparisons were madeby two-way ANOVA. Additional analyses were made by comparing changes inrenal perfusion pressure and changes in mean arterial pressure from therespective pre-infusion values (i.e., baselines) among BAA-NP, BUA-NP,and BNP using one-way ANOVA.

The result using BAA-NP are set forth in FIGS. 3-29, and the resultsusing BUA-NP are set forth in FIGS. 30-56. Comparisons with human BNP onmean arterial pressure and renal perfusion pressure are set forth inFIGS. 57 and 58. Additional analyses comparing changes in renalperfusion pressure from baseline and changes in mean arterial pressurefrom baseline among BAA-NP, BUA-NP, and BNP are set forth in FIGS. 59 to62. Comparisons between BAA-NP and BUA-NP are set forth in FIGS. 63 to73.

Both BAA-NP and BUA-NP significantly increased plasma cGMP, urinary cGMPexcretion, net renal generation of cGMP, urine flow, and urinary sodiumexcretion. Both proximal and distal fractional reabsorption of sodiumwere significantly reduced. Urinary potassium excretion was alsosignificantly increased. Glomerular filtration rate was preserved. Renalblood flow was significantly enhanced, and renal vascular resistance wassignificantly reduced. With BUA-NP, renal perfusion pressure was alsopreserved.

Both BAA-NP and BUA-NP significantly reduced pulmonary capillarypressure, right atrial pressure, and pulmonary arterial pressure.Cardiac output was preserved. A mild decrease in mean arterial pressurewas detected. Hemoconcentration was observed, as assessed by asignificant increase in hematocrit. Plasma ANP and CNP immunreactivitieswere significantly increased. With BAA-NP, urinary excretion of ANP andCNP were also significantly increased. Plasma renin activity,angiotensin II, and aldosterone were significantly suppressed by BAA-NP.

When tested at an equimolar concentration, human BNP significantlyreduced mean arterial pressure and renal perfusion pressure. Betweengroup differences were not detected among BNP, BAA-NP, and BUA-NP.

When additional analyses were performed focusing on changes frombaseline values, significantly greater decreases in renal perfusionpressure were observed with BNP, as compared to BAA-NP or BUA-NP, bothat 30 minutes and at 60 minutes of infusion. In other words, both BAA-NPand BUA-NP, as compared to BNP, preserved renal perfusion pressure. Withregard to changes in mean arterial pressure, a significantly greaterreduction of mean arterial pressure from baseline was observed with BNP,as compared to BAA-NP or BUA-NP, at 30 minutes of infusion. This greaterdecrease in mean arterial pressure from baseline was also observed at 60minutes of infusion with BNP versus BAA-NP. When the two natriureticpolypeptides, BUA-NP and BAA-NP, were compared, BUA-NP was observed toresult in greater activation of cGMP, enhanced natriuresis and diuresis,and greater reductions in proximal and distal fractional reabsorption ofsodium. Thus, fusion of the N-terminal tetrapeptide, TAPR, from theN-terminus of urodilatin to produce BUA-NP further enhanced the renalactions of BAA-NP. Overall, these results indicate that the engineeredchanges to the N- and C-termini of BNP provided herein can result ineffective natriuretic polypeptides with enhanced pharmacologic profiles.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. (canceled)
 2. A method for increasing natriuretic activity within amammal, wherein said method comprises administering, to said mammal, acomposition comprising a polypeptide under conditions whereinnatriuretic activity within said mammal is increased and underconditions wherein the renal perfusion pressure of said mammal ismaintained to a greater extent than the renal perfusion pressuremaintained in a control mammal administered human BNP, wherein saidpolypeptide is less than 44 amino acid residues in length and comprises,in an order from amino terminus to carboxy terminus: (a) the sequenceset forth in SEQ ID NO:6 or the sequence set forth in SEQ ID NO:6 withno more than two additions, subtractions, or substitutions, (b) thesequence set forth in SEQ ID NO:2 or the sequence set forth in SEQ IDNO:2 with no more than one addition, subtraction, or substitution, and(c) the sequence set forth in SEQ ID NO:7 or the sequence set forth inSEQ ID NO:7 with no more than two additions, subtractions, orsubstitutions.
 3. The method of claim 2, wherein said compositioncomprises a pharmaceutically acceptable carrier.
 4. The method of claim2, wherein said polypeptide comprises the sequence set forth in SEQ IDNO:6, the sequence set forth in SEQ ID NO:2, and the sequence set forthin SEQ ID NO:7.
 5. The method of claim 2, wherein said polypeptide is asubstantially pure polypeptide.
 6. The method of claim 2, wherein saidpolypeptide comprises the sequence set forth in SEQ ID NO:6.
 7. Themethod of claim 2, wherein said polypeptide comprises the sequence setforth in SEQ ID NO:6 with no more than two additions, subtractions, orsubstitutions.
 8. The method of claim 2, wherein said polypeptidecomprises the sequence set forth in SEQ ID NO:6 with one addition,subtraction, or substitution.
 9. The method of claim 2, wherein saidpolypeptide comprises the sequence set forth in SEQ ID NO:2.
 10. Themethod of claim 2, wherein said polypeptide comprises the sequence setforth in SEQ ID NO:2 with one addition, subtraction, or substitution.11. The method of claim 2, wherein said polypeptide comprises thesequence set forth in SEQ ID NO:7.
 12. The method of claim 2, whereinsaid polypeptide comprises the sequence set forth in SEQ ID NO:7 with nomore than two additions, subtractions, or substitutions.
 13. The methodof claim 2, wherein said polypeptide comprises the sequence set forth inSEQ ID NO:7 with one addition, subtraction, or substitution.
 14. Themethod of claim 2, wherein said polypeptide, when administered to amammal, induces a decrease in mean arterial pressure to a lesser extentthan that induced by human BNP, when said human BNP is administered to amammal.